Vaccinia viruses belong to the family of poxviruses. Certain strains of vaccinia viruses have been used for many years as live vaccine to immunize against smallpox, for example the Elstree strain of the Lister Institute in the UK. Because of the complications which may derive from the vaccination (compare Schar, Zeitschr. fur Praventivmedizin 18, 41-44 [1973]), and since the declaration in 1980 by the WHO that smallpox had been eradicated nowadays only people at high risk are vaccinated against smallpox.
Vaccinia viruses have also been used recently as vectors for foreign antigens (Smith et al., Biotechnology and Genetic Engineering Reviews 2. 383-407 [1984]). This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia viruses. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (compare European Patent Applications with the publication Nos. 83,286 [published on Jul. 6, 1983] and 110,385 [published on Jun. 13, 1984]). The recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infections which are caused by these antigens or, on the other hand, for the preparation of heterologous proteins in eukaryotic cells. Most of the recombinant vaccinia viruses described in the literature are based on the WR strain. On the other hand, it is known that this strain has a high neurovirulence and is thus poorly suited for use in humans and animals (Morita et al., Vaccine 5, 65-70 [1987]).
However, strains of viruses specially cultured to avoid undesired side effects have been known for a long time. Thus, it has been possible, by serial passages of the Ankara strain of vaccinia virus (CVA) on chicken fibroblasts, to culture a modified vaccinia virus Ankara (MVA) (Swiss Patent No. 568,392). This modified vaccinia virus Ankara has only low virulence, that is to say it is followed by no side effects when used for vaccination. Hence it is particularly suitable for the initial vaccination of immunocompromised subjects. The excellent properties of the MVA strain have been demonstrated in a large number of clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167. 375-390 [1978], Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 [1974]).
Hence, a possible way to introduce foreign genes by DNA recombination into the MVA strain of vaccinia virus has been sought. Since the intention was not to alter the genome of the MVA except at the recombination site, it was necessary to use a method which complied with this requirement. Since the thymidine kinase gene (TK gene) of MVA is not of fundamental importance for the life cycle of the virus, the foreign DNA sequence was recombined in the DNA of the vaccinia TK gene.